Learning from extremophilic fungi: proteomics studies of the

Stress resistance and adaptation are crucial factors determining an organisms survival to life threatening environmental conditions. Black fungi, a group of fungi whose prominent characteristic is a dark coloration given by melanin, have an extraordinary ability to survive harsh living conditions, which makes them the most stress resistant Eukaryotes known to date. Hence, they can be considered as good models for studies of extremophilia and extremotolerance and of adaptation. For the present project, two strains of extremotolerant black fungi i.e. the wild type and the natural non-melanized mutant of the species Knufia chersonesos will be used as model organisms to elucidate the cell processes which lie behind ozone stress tolerance, by proteomic methods and to ultimately detect proteins with key roles in counteracting stress insults. The two strains indeed display a remarkable natural aptitude to tolerate ozone rates beyond the values considered harmful for plant and animal tissues and will therefore aid the study of the dynamics of the oxidative stress defense triggered by the exposure to ozone. The data obtained will allow shedding light on the oxidative stress, a condition associated with several physiological and also pathological conditions. The understanding of the basis for tolerance in adapted species will possibly help accomplishing a second objective of the present project: the finding of novel proteins with potential biotechnological and cosmetic applications. Protein stability and degradation tests will be carried out and special focus will be dedicated to enzymes involved in degradation of the biodegradable polymer PBAT, better known as Ecoflex. Ozone treatment and Ecoflex-mediated induction of fungi grown in liquid culture will be performed by selecting a number of time points, in order to evaluate the effects of short- and long-term exposure. Shotgun quantitative proteomics approaches for simultaneous detection of changes in protein abundance and identification, will be applied to the analysis of both whole-cell proteome and secreted proteins. The realization of this project will rely on an interdisciplinary team of experts in proteomics and microbiologists, which include national (BOKU VIBT, BOKU IFA Tulln and VET-MED) and international partners (the University of Florida and the University of Geneva). Their contribute to the establishment of cysTMTRAQ and fermentations work-flow applied for the first time to the study of black fungi will bring innovation into the black fungi research field and will allow to get a deeper insight into the protein regulatory pathways at the base of stress resistance. The obtained results will additionally be of impact for the biotech and, for example as skin protectant, in cosmetic industry.

The goal of this project was to get a deeper understanding of the ecology of fungal extremophiles by shedding light on the mechanisms responsible for their stress tolerance, as well as to search for novel enzymes and compounds with potential biotechnological applications. The project consisted of three major research lines revolving around the rock black fungus Knufia chersonesos (syn. K. petricola), used as model organism: 1_ Screening of enzymes involved in the breakdown of synthetic polymers; 2_ Study of the stress tolerance and limits for life: effects of microgravity on fungal ecophysiology and 3_ Study of the oxidative stress response. A systems biology approach, mostly involving shot-gun proteomics, was applied to detect qualitative and quantitative changes in protein abundance ratios upon stress simulation and control conditions, with the ultimate goal to identify candidate proteins with key roles in polymer degradation and stress survival. Bioinformatics analyses were performed to gain information about protein identities and functionalities. Being K. chersonesos an emerging model organism, whole genome sequencing and homology-based genome ab initio translation had to be performed prior to the proteome screening. The datasets were deposited in the online public databases NCBI and ProteomeXchange, respectively. This project has given its contribution to the advancement of the knowledge on the physiology and systems biology of black fungi by the investigation of the mechanisms of stress adaptation at the proteome level. Moreover, the screening of the fungal secretome has highlighted, for the first time, K. chersonesos aptitude to degrade synthetic polymeric material (i.e. PBAT) by means of extracellular enzymes of potential biotechnological use. This research has culminated in the identification of protein candidates to be further investigated through biochemical characterization, which will aid a deeper comprehension of protein adaptations and functionalities in view of their application. The study of the fungus response to microgravity conditions on the other hand, has allowed to get a deeper insight into the evolution of extremophily and the limits for life beyond planet Earth. The project has therefore proved to be of relevance for areas of research such as astrobiology, in addition to biotechnology and microbiology. Furthermore, the development of experimental workflows and methods for fungal cultivation (i.e. upon exposure to polymers, microgravity and ozone) will pave the way for more of similar studies to come.